Mass spectrometry is an instrumental analysis technique whereby sample molecules are ionized and then separated in accordance with the mass/charge ratio (m/z) for detection. Using this technique, qualitative analysis can be performed based on the resultant mass spectrum, and quantitative analysis can be performed based on ion quantities.
The mass spectrometer (“MS”) used for such a measurement of molecular mass roughly consists of an ionization unit (ion source) for ionizing a sample, an analyzer for separating ions in accordance with the mass/charge ratio m/z (m: mass, and z: charge number), a detection unit (detector) for detecting separated ions, and a data analysis unit.
When subjecting sample molecules to mass spectrometry using the aforementioned mass spectrometer, the mass spectrometer must be calibrated prior to measurement. Specifically, since errors might be introduced into the measurement by the mass spectrometer due to factors such as temperature changes, voltage accuracies, and electric circuit noise, a calibration procedure must be carried out prior to the start of measurement. In the calibration procedure, the chromatograph or the like is removed from the mass spectrometer, and a predetermined mass-calibration standard substance is introduced into the mass spectrometer so as to obtain an observed mass value. The observed mass value is compared with a known theoretical mass value, and the apparatus is adjusted such that no systematic error occurs in mass values (a calibration procedure according to the external standard method).
If an even higher accuracy of mass values is to be obtained, an additional calibration procedure must be performed, whereby a known substance is mixed in the sample and its mass is measured, and the actual measurement value is adjusted based on the mass value (a calibration procedure according to the internal standard method).
In general, identification of biopolymers, such as peptides or proteins, using a mass spectrometer (including the tandem mass spectrometer) involves a procedure referred to as a database search (or a library search). In this procedure, the observed mass value of an unknown sample molecule obtained by mass spectrometry is searched for by matching with a database (library) in which the primary structures or sequences of approximately 100,000 kinds of molecules are stored. In an expected reference (standard) spectrum calculated based on the structure information, molecules with a spectrum similar to that of the unknown molecule under investigation are allocated scores and selected. Candidate molecules are thus narrowed and listed, thereby eventually identifying the unknown sample molecule.
However, the above-described mass spectrometer calibration procedure is very troublesome work, requires much adjustment time, and is primarily responsible for the drop in work efficiency caused by the conventional mass measurement operation. Namely, it has been impossible to carry out a measurement operation with high efficiency based on a continuous operation of the mass spectrometer (without calibration). Further, in a measurement system employing a plurality of mass spectrometers, it has been extremely difficult to achieve uniform accuracy and reliability in the individual apparatuses even if they are calibrated individually according to the external standard.
In the case of the external standard calibration, it has been impossible, using the conventional process of database search as described above, to eliminate from the measurement data the influence of erroneous measurement in the mass spectrometer produced by influences of the external environment. Particularly, even those measurement errors due to subtle temperature changes (on the order of 0.2° C.) in the measurement environment could not be ignored in some cases.
Furthermore, when a complex biopolymer mixture is measured by the conventional internal standard calibration method, the internal standard substance and the ion signals from the sample are superposed, which prevents ion analysis. Thus, it has been difficult to select the type or concentration of the substance that is put into the sample as the internal standard. In order to achieve high mass accuracy for a wide range of masses, it has been necessary to introduce a number of internal standard substances.
Also, human confirmation of each identification result has been necessary, as the identification reliability has been low. Recent progress in mass spectrometry, however, has made direct analysis of increasingly more complex biopolymer mixtures possible. This has resulted in huge volumes of data that could not possibly be individually confirmed by the human eyes. Therefore, there has been a need to develop a highly reliable automatic identification technique for the analysis of complex biopolymer mixtures.